The goal of these studies is to understand the molecular mechanism of homologous genetic recombination in human cells. Homologous recombination plays an integral role in maintaining genomic stability and is an important mechanism for the repair of chromosomal double-strand breaks. This study focuses on the functional organization of the human Rad52 protein (HsRad52) as well as the functional relationships between HsRad52 and other human recombination proteins. HsRad52 mediates the catalytic recombination activity of HsRad51 via a mechanism that has yet to be defined in molecular terms. HsRad52 binds both single and double-stranded DNA, interacts specifically with HsRad52 and HsRPA, and forms both ringshaped oligomers as well as higher-order assemblies of these rings. We have recently identified a domain within HsRad52 that specifically regulates formation of these higher-order structures and have demonstrated that these structures are relevant to protein function. In these studies we will define residues within HsRad52 that are important for each of its biochemical activities, and will carry out studies designed to understand the importance of each of these activities with regard to their role in mediating HsRad51-catalyzed strand exchange. An additional aim of our work is to define the molecular nature of the functional relationships between HsRad52 and the HsRad51 paralog proteins. For this work we have initiated RNA interference studies using human cell lines. RNAi-mediated knock down of endogenous gene products allows us to assess the function of mutant versions of the corresponding transgene for which we have mechanistic information from biochemical studies of that mutant protein. This combination of methods will provide important insights into specific biochemical aspects of HsRad52 and HsRad51 paralog proteins that are required for optimal function of the homologous recombination pathway in vivo. Understanding the molecular mechanistic principles of homologous recombination in humans has far-reaching effects for creating novel proteins with desired properties that may be used for prevention of diseases resulting from genomic instability and for beneficial genetic manipulation. [unreadable] [unreadable]